qPCR experiment design

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Presentation transcript:

qPCR experiment design

Experiment Absolute qPCR Relative qPCR Standard 1 Standard 2 NTC Sample B Standard 5 Standard 6 Ref1 St 1 Ref1 St 2 Ref1 St 3 Ref1 St 4 Ref2 St 1 Ref2 St 2 Ref2 St 3 Ref2 St 4 Ref3 St 1 Ref3 St 2 Ref3 St 3 Ref3 St 4 GOI St 1 GOI St 2 GOI St 3 GOI St 4 Ref1 Sample 1 Ref2 Sample 1 Ref3 Sample 1 GOI Sample 1 Ref1 Sample 2 Ref2 Sample 2 Ref3 Sample 2 GOI Sample 2

Target Target = target gene = a gene you will detect with a certain primer pair primers are designed to anneal on their target gene If primers also anneal on another gene, it will cause off-target (non-specific) amplification Please write down what are the targets in this experiment: 1. 2. 3. 4. 5.

where else on the plate will it be? Please use colours to mark reactions (plate wells) with the same target Absolute Relative for example: Ref gene 1 where else on the plate will it be? plate layout 1 2 3 4 5 6 7 8 9 10 11 12 A B C D E F G H Reference gene 1

n of reactions for Absolute qPCR n of reactions for Relative qPCR Please count the number of reactions (plate wells) with the same target Target gene name n of reactions for Absolute qPCR n of reactions for Relative qPCR

Final concentration of each primer in the reaction should be 125 nM Please write down premix composition for each target Absolute Relative premix x1 x 2x master mix 10 ul 100 uM primer Fw 0,025 ul 100 uM primer Re template 5 ul MQ 4,95 ul You are welcome to dilute your primers stocks 20 or mote times to make pipetting more convenient. If you do so, please recalculate primer and MQ volumes you will add to the reactions. Final concentration of each primer in the reaction should be 125 nM

Please write down what are the templates used in this experiment: Template = DNA you add to the PCR (e.g. genomic DNA, cDNA, plasmid etc.) Please write down what are the templates used in this experiment: 1. 2. 3. 4. 5. …

Please mark reactions with exactly the same template (use symbols or text. sic! in this case different dilutions do not count as exactly the same template) Absolute Relative plate layout 1 2 3 4 5 6 7 8 9 10 11 12 A B C D E F G H

n of reactions for Absolute qPCR n of reactions for Relative qPCR Please count the number of reactions with the exactly the same template template name n of reactions for Absolute qPCR n of reactions for Relative qPCR

n of reactions for Absolute qPCR n of reactions for Relative qPCR Please calculate the total volume of each template you will need template name n of reactions for Absolute qPCR Volume in uL n of reactions for Relative qPCR

What is the dilution factor you are going to use? Absolute qPCR What dilutions you will make for in this experiment and is the purpose of these dilutions? What is the dilution factor you are going to use? Please note, that for the sake of time you will not dilute the sample with unknown concentration for this experiment. Please remind yourself, why would it be beneficial to dilute it if running this type of experiment for the very first time?

Please write ul of each component for each dilution and mention which components are used for what dilution Absolute qPCR sample to be diluted ul of sample ul of MQ Standard curve dilution 1 Standard curve dilution 2 Standard curve dilution 3 Standard curve dilution 4 Standard curve dilution 5 Standard curve dilution 6

Relative qPCR What dilutions you will make for this experiment? What is the purpose of these dilutions? What is the dilution factor you are going to use for efficiency curves? What will be the composition of your DNA pool for primer efficiency and why? How much are you going to dilute your samples and why?

Please write ul of each component for each dilution and mention which components are used for what dilution Relative qPCR sample(s) ul of sample(s) ul of MQ DNA pool DNA pool dilution 1 DNA pool dilution 2 DNA pool dilution 3 DNA pool dilution 4 sample 1 sample 2

Protocol (or PCR program) what happens here? 1. 95 oC 7 minutes what happens here? why not more or less? 2. 95 oC 10 seconds 3. 60 oC 30 seconds 40 repeats (acquisition) what happens here? what is this for? why is it at this step? 4. 95 oC 1 minute what is this for? 5. 60-95 oC acquisition after each 0.5 oC change what happens here? what is this for?